Synovial joints are essential for skeletal function and quality of life and much is known about their anatomical organization, distinct tissues and susceptibility to common pathologies including age-dependent osteoarthritis. In contrast, very little is known about their developmental biology. Were such information available, it could be used directly or in combination with bioengineering tools to create new joint repair and regenerative strategies such as engineered stem cells with specific joint tissue-formation capacity. This project started five years ago to fill such glaring gaps in information. In the limbs, synovial joint formation initiates with the appearance of mesenchymal cells (collectively called the interzone) at each prospective joint site flanked by cartilaginous long bone anlaga. However, it had long remained unknown how the interzone cells acquire their essential mesenchymal character, whether they serve as a critical but transient joint demarcation point, and/or whether they actually produce joint tissues over time. To address these fundamental questions, we genetically traced and tracked the interzone cells prenatally and postnatally in mice. Strikingly, we found that the cells are not at all transient but produce joint tissues and only joint tissues and thus, constitute a specialized cohort of multipotent progenitor cells endowed with joint formation capacity. We showed that at early stages the cells exhibit strong Wnt/-catenin signaling. As joint development progresses, signaling dwindled but remained strong in the superficial zone of articular cartilage (critical for joint function and characterized by a unique fibro-cartilaginous phenotype). Indeed, conditional -catenin ablation caused a near loss of that zone. Additional Preliminary Data now indicate that the Wnt/-catenin pathway does not act alone, but cooperates with the retinoid pathway and nuclear retinoic acid receptors (RARs) to sustain and regulate joint formation. The data lead to the central hypothesis for this competitive renewal application that the retinoid and Wnt/-catenin signaling pathways establish the multipotent mesenchymal character of early interzone cells and are then topographically modulated to permit formation of distinct joint tissues. Maintenance of signaling activity would allow interzone cells to produce fibrous and fibro-cartilaginous joint tissues, while a drop in signaling would allow formation of articular cartilage. Our Aims are: (1) to further characterize the roles of retinoid signaling in interzone function and joint formation; (2) to determine molecular mechanisms by which the two signaling pathways interact to regulate interzone cell function and joint formation; and (3) to determine interzone cells' plasticity and differentiation potentials and regulation by retinoid and Wnt/-catenin signaling. The work proposed in this continuation proposal combines experimental lines on basic regulatory mechanisms with lines testing plasticity, transplantability and developmental potentials of interzone cells. It will thus continue to produce information of fundamental value to basic biologic knowledge and will also significant relevance for translation medicine and creation of future novel joint repair and regeneration therapies.